An ASO for Eamonn

Charles River scientists are developing the first ever antisense oligonucleotide (ASO) for a child with the inherited disorder Dup15q

Sam Quigley remembers distinctly the first time he looked down at his newborn son Eamonn and suspected something was wrong. He and his wife were in Italy when they noticed their 4-month-old kept doing "funny things with his arms and legs." Quigley initially thought it was due to constipation, but the jerky motions were actually infantile spasms, a severe form of epilepsy. In Eamonn's case, the spasms were triggered by a rare genetic disease known as Dup15q syndrome that geneticists finally zeroed in on when Eamonn was 8 months old. Other common symptoms of Dup15q include decreased muscle tone, motor delays and variable intellectual disability. Many children develop autism, too.

"As a parent, news like this is world-shattering," said Quigley. "I mean, you plan for all these things when your child is born, you think he is going to grow up to be an astronaut, and just like that, those dreams sort of crumble."

Indeed, the Quigleys suddenly found themselves focusing on stuff they never in a million years thought they needed to know as new parents, like finding the best specialists or dealing with difficult health insurers. Fortunately, they linked up early on with an excellent pediatric neurologist in Italy who had trained at University of California, and their luck with doctors and physical therapists continued after they moved back to the US and settled in New York City. "Our son, who is now 9, has gotten very good therapy from a very young age," says Quigley. "You can see it in him. He works really hard, partly because everything is hard. He has epilepsy, intellectual disability, all his muscles are weak. He has difficulty managing his movements and he has poor motor control. But he loves succeeding, and if he does succeed, he gets a goofy grin."

About two years ago, Quigley, one of the early developers of the financial services platform Square, launched Kicho Inc. to find a cure for Dup15q, focusing first on a novel antisense drug for Dup15q syndrome. Kicho, a name inspired by Eamonn's middle name Kichizio, is partnering with Charles River Laboratories to design, develop and test the ASO-based therapy. More than 100 Charles River scientists at seven different sites, from Kuopio, Finland to Reno, Nevada, are working to make it happen.

"Drug discovery takes a lot of intellectual input from many disciplines," David Fischer, Chief Technology Officer Discovery at Charles River, says. "Together with a number of strategic partners, such as Fios Genomics and bit.bio, our talented researchers from both Discovery and Safety Assessment have designed, run and analyzed molecules and experiments to drive this program forward in the most efficient manner."

Carrie Howell, Executive Director of the Dup15q Alliance, works with many families dealing with this rare genetic condition. While her organization is not directly involved in the development of Eamonn's ASO, she knows how important it is to find effective treatments for rare diseases. "Our parents are so weary because they want to give their Dup15q loved one the best possible life," says Howell. "They fear SUDEP [Sudden Unexpected Death in Epilepsy] and losing their child to a seizure. They endure sleepless nights of watching seizure monitors or having their loved one endure the restless phases of Dup15q. Chronic constipation, major behavioral shifts, loss of verbal skills, and issues that can arise, likely due to hypotonia, are the top concerns parents share when they call the Dup15q Alliance. The parents are desperate for the hope that something like this ASO could bring to their child."

What is Dup15q syndrome?

Dup15q syndrome and two other rare genetic diseases, Prader-Willi syndrome and Angelman syndrome, result from the loss of function or overexpression of at least one imprinted gene found on the 15q11-q13 locus or region of chromosome 15. Dup15q Syndrome got its name because it has a duplication on a region of the long ("q") arm of Chromosome 15.

Genomic imprinting refers to the process by which certain genes are 'branded' with the parent of origin. Normally, when sperm and eggs are made, epigenetic markers inherited from the parent or accumulated in life are removed in the offspring, but in genes that undergo genomic imprinting, new markers are added that identify the gene as coming from either the mother or the father. These new markers change gene expression, resulting in the imprinted copy of the gene being turned off and the other copy being turned on. For instance, if the copy of the gene inherited from the father is imprinted, it is silenced and only the copy of the gene from the mother expressed.

Eamonn has an isodicentric duplication-science-speak for having an extra maternally-derived copy of a certain region of chromosome 15. Isodicentric duplication is the most common form of Dup15q syndrome. While there are multiple genes of interest located in this region, evidence suggests that the ubiquitin-protein E3A ligase (UBE3A) gene is the predominant molecular cause driving Dup15q syndrome. The preclinical work being conducted at Charles River is focused on reducing UBE3A expression levels with an antisense oligonucleotide (ASO) drug. The hope is that reducing gene expression can reduce the severity of Eamonn's symptoms. If it pans out, it will be the first successful ASO drug for a child with Dup15q.

"I get a lot of energy and feel a sense of accomplishment knowing we are close to having a drug that is hopefully going to be delivered to a little boy suffering from this disease," says Associate Science Director Roxana Redis, PhD, who oversees the ASO Discovery Group at Charles River's Leiden site in the Netherlands. "That is very inspiring to me, the most inspiring part of my job."

Finding the right antisense drug

To understand how ASOs work, it is helpful to first understand how genes work. Genes encode for proteins, which in turn dictate cellular functions. The genetic information is passed on to proteins by messenger RNA (mRNA). In Dup15q, the usual two copies of chromosome 15 plus two duplicated copies of a segment of chromosome 15, deliver much more mRNA for those genes on the Dup15 sequence than the cell normally makes. This increased gene expression disrupts normal cellular development, underlying the characteristic features of Dup15q.

For many genetic diseases, ASOs represent a way of dealing with genes that are mutated or not expressed at the right level. ASOs are chemically engineered drugs designed to specifically target mRNAs. Some ASOs cause skipping to correct for mutations, but the therapy being developed for Eamonn aims to restore the right levels of one of the genes causing the disease, or part of the symptoms.

ASOs can act through many mechanisms. Some modulate RNA expression levels or modify RNA transcripts. Sometimes they reduce translation of the gene. The ASO being developed for Eamonn is designed to cause the degradation of the target mRNA, says Maria Blanca Torroba, PhD, a Senior Scientist in Leiden who led the project to develop the Dup15q drug.

One of the earliest ASOs to reach the market, for spinal muscular atrophy, helped most patients to walk without assistance. Today there are 15 ASOs approved for use in the US. Many other ASOs have been developed for single patients and tested in n-of-1 trials.

The hunt for an ASO for Eamonn's form of Dup15q began at Charles River's Leiden site in 2021. In collaboration with Fios Genomics Charles River's bioinformatics partner, and ASO design consultant, Jim McSwiggen, more than 1,000 ASO sequences out of 60,000 were generated with the potential of targeting and reducing UBE3A mRNA in both rats and humans. Next, they used skin fibroblasts to design an ideal cell candidate, and began screening 200 oligonucleotides, ranking them based on their predictive value for success. From there it was a long and arduous process of narrowing down the list of candidates based on a long list of assays and data points.

The in vitro and in vivo tests included immunotoxicity tests, which detect if an ASO generates an unwanted immune response, testing of cross-reactivity in rat fibroblasts and more complex in vitro in Dup15q and control neurons to derisk the candidates and elicit as much information as possible in order to pick the winning ASO. The first rodent screen, in mice, included 24 candidates. Later 10 candidates were tested in rats and one in a mouse to determine the highest and safest dose of the compound, and how well it circulates in the CNS and distributes itself.

Torroba said in the beginning they hoped to create an ASO unique to Eamonn, to allow for faster clinical approval. "Usually the patient-specific therapies are more easily approved, but the sequencing data from the patient did not pass the criteria needed based on the [single nucleotide polymorphism] found, so we took a more general approach trying to knock down the gene scanning ASO sequences across the entire reference sequence," said Torroba. "This therapy, in principle, would be applicable to many Dup15q patients where UBE3A KD might yield therapeutic benefits indeed."

At the end of the day the 154th ASO out of 200 rose to the top because it worked in skin fibroblast, human neurons, and both mice and rats and appeared to have a strong safety profile. Mice even tolerated it in high doses. More work is needed to optimize the drug to improve how well the compound circulates in the brain, and importantly, where it accumulates. If the findings are good, the ASO could move to the critical next step, the IND-enabling phase that is required for an Investigational New Drug submission to the Food and Drug Administration. The studies include in vivo safety/tolerability studies and the manufacture of GMP-grade drug.

Quigley said their academic collaborators at the University of North Carolina also recently provided some positive data in their mouse model of Dup15q, which displayed some distinct phenotypes. "We demonstrated that our ASO completely corrects the most serious one-a SUDEP-like seizure that is fata," he said, adding that he will be announcing results at the Dup15q Alliance/Angelman Syndrome Foundation Science Symposium this month.

Michael V. Templin, PhD DABT, part of Charles River's Scientific Advisory Services and a member of the Development Team working on Eamonn's drug, said in any ASO project you face two essential questions: Can you design an ASO for a precise action, and what are the challenges and opportunities. "Because Dup15q is a duplication case, where patients unfortunately produce excess protein, the intent is not to eliminate it but to bring it to a normal range," said Templin. "That is an added twist to this case because it is always easier to eliminate as compared to moderate."

An ASO project that took an integrated approach

Designing and developing ASOs, once a rarity, is now a common focus of Charles River's rare disease work. Still, the Dup15q project represented a first of sorts for the company because almost all of the research was done inhouse. Templin believes this integrated approach, which has joined together the Discovery and Safety teams at Charles River, has made a big difference in moving complex projects forward more efficiently. "We found places to streamline, we found places we could work closer with discovery and safety assessment and hand programs off with the least amount of cost impact and maximum use of time," he said.

Redis agrees. "A lot of companies are under the impression that they are better off choosing different places to work on the different stages of their project, when in reality the time it takes to invest in all these different entities won't make it faster or cheaper for them."

There is still a lot of research road ahead, but Sam is hopeful. "A project like this has lots of ups and downs, but I'm really excited by this latest round of data. It validates the course we're taking and gives me hope that we'll be in the clinic soon," says Quigley. "There's still plenty of work ahead but I am going to do everything possible to make it happen. As a dad, how could I do anything else?"

Howell says the limitless power of a caregiver who sees the daily struggles that can come with a rare disease like Dup15q syndrome drives groups like theirs to continue in the fight for therapeutics for their patients. "It is nothing short of heroic to see a parent use their resources and abilities to bring to fruition something like an ASO."

With the exception of the lab shot, all pictures courtesy of Sam Quigley. Top right, Eamonn with a portable EEG device, middle right and bottom left, Eamonn with his dad Sam.

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